Paul Kirkham

Inhibition of PI3Kdelta restores glucocorticoid function in smoking-induced airway inflammation in mice.

RATIONALE There is an increasing prevalence of reduced responsiveness to glucocorticoid therapy in severe asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanism of this remains unknown. Recent studies have shown that histone deacetylase activity, which is critical to glucocorticoid function, is altered by oxidant stress and may be involved in the development of glucocorticoid insensitivity. OBJECTIVES To determine the role of phosphoinositol-3-kinase (PI3K) in the development of cigarette smoke-induced glucocorticoid insensitivity. METHODS Wild-type, PI3Kgamma knock-out and PI3Kdelta kinase dead knock-in transgenic mice were used in a model of cigarette smoke-induced glucocorticoid insensitivity. Peripheral lung tissue was obtained from six healthy nonsmokers, nine smokers with normal lung function, and eight patients with COPD. MEASUREMENTS AND MAIN RESULTS In vitro oxidative stress activates PI3K and induced a relative glucocorticoid resistance, which is restored by PI3K inhibition. In vivo, cigarette smoke exposure in mice increased tyrosine nitration of histone deacetylase 2 in the lung, correlating with reduced histone deacetylase 2 activity and reduced glucocorticoid function. Histone deacetylase 2 activity and the antiinflammatory effects of glucocorticoids were restored in PI3Kdelta kinase dead knock-in but not PI3Kgamma knock-out smoke-exposed mice compared with wild type mice, correlating with reduced histone deacetylase 2 tyrosine nitration. Glucocorticoid receptor expression was significantly reduced in smoke-exposed mice, in smokers with normal lung function, and in patients with COPD. CONCLUSIONS These data show that therapeutic inhibition of PI3Kdelta may restore glucocorticoid function in oxidative stress-induced glucocorticoid insensitivity.

Curcumin Restores Corticosteroid Function in Monocytes Exposed to Oxidants by Maintaining HDAC2

Oxidative stress as a result of cigarette smoking is an important etiologic factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. Histone deacetylase-2 (HDAC2), a critical component of the corticosteroid anti-inflammatory action, is impaired in lungs of patients with COPD and correlates with disease severity. We demonstrate here that curcumin (diferuloylmethane), a dietary polyphenol, at nanomolar concentrations specifically restores cigarette smoke extract (CSE)- or oxidative stress-impaired HDAC2 activity and corticosteroid efficacy in vitro with an EC(50) of approximately 30 nM and 200 nM, respectively. CSE caused a reduction in HDAC2 protein expression that was restored by curcumin. This decrease in HDAC2 protein expression was reversed by curcumin even in the presence of cycloheximide, a protein synthesis inhibitor. The proteasomal inhibitor, MG132, also blocked CSE-induced HDAC2 degradation, increasing the levels of ubiquitinated HDAC2. Biochemical and gene chip analysis indicated that curcumin at concentrations up to 1 muM propagates its effect via antioxidant-independent mechanisms associated with the phosphorylation-ubiquitin-proteasome pathway. Thus curcumin acts at a post-translational level by maintaining both HDAC2 activity and expression, thereby reversing steroid insensitivity induced by either CSE or oxidative stress in monocytes. Curcumin may therefore have potential to reverse steroid resistance, which is common in patients with COPD and asthma.

Cigarette smoke triggers macrophage adhesion and activation: role of lipid peroxidation products and scavenger receptor.

Pulmonary emphysema in chronic obstructive pulmonary disease (COPD) is characterized by the destruction of the alveolar walls leading to permanent enlargement of distal respiratory air spaces. A major causal factor is cigarette smoking, which produces conditions of chronic oxidative stress within the lungs. At a cellular level, increased macrophage accumulation and retention within the alveolar interstitial spaces is pivotal to the development of emphysema. To date it has been unclear as to the underlying mechanisms relating chronic oxidative stress to macrophage accumulation and retention. Our study was initiated to ascertain the role of modification of extracellular matrix proteins with cigarette smoke and products of lipid peroxidation on macrophage adhesion and activation. Increased numbers of macrophages were seen adhering to cigarette smoke-modified collagen IV as compared to unmodified collagen, where little or no adherent macrophages were observed. Similar observations were made when collagen was modified with either acrolein or 4-hydroxy-2-nonenal. Adhesion could be blocked with either fucoidan or a monoclonal antibody against the Type A macrophage scavenger receptor. Also, modified collagen triggered both oxidative burst and MCP-1 release in macrophages. These results, therefore, highlight a potential mechanism by which oxidative stress through the production of reactive carbonyls promotes macrophage accumulation, retention, and activation, independently of other proinflammatory stimuli. The implications of this for the development of emphysema in COPD are discussed.

Oxidative stress–induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease

Background Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology. Objective We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. Methods Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. Results Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release. Conclusions Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

LPS induced inflammatory responses in human peripheral blood mononuclear cells is mediated through NOX4 and Giα dependent PI-3kinase signalling

COPD is a disease of innate immunity and bacterial infections are a dominant cause of exacerbations in the later stages resulting in poor health and high mortality. The pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) is sensed by immune cells through activation of the toll-like receptor 4 (TLR4). This leads to the activation of NADPH oxidase (NOX) and NF-κB which together drive COPD inflammation. In this study we show in human PBMCs that LPS stimulated proinflammatory cytokine release (CXCL8 and IL6) was inhibited by approximately 50% by the broad specificity phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. Our results also demonstrate that activation of PI3K following LPS stimulation is mediated by a NOX4 dependent mechanism releasing endogenous H2O2, as the NOX4 inhibitor apocynin blocked LPS induced AKT phosphorylation. Moreover, LPS-induced PI3K activation was inhibited by the anti-oxidant N-acetylcysteine in a concentration dependent manner (IC50 ~100 μM). In addition, our data demonstrated that inhibition of small G proteins, by pre-treatment with pertussis toxin, inhibited LPS-induced AKT phosphorylation. Furthermore, the G-protein inhibitors pertussis toxin and mastoparan both inhibited LPS-induced CXCL8 and IL-6 release by approximately 50%. Together, these data indicate there is a mechanism in human PBMCs where TLR4 activation by LPS leads to ROS generation through NOX4 and activation of the PI3K pathway. This effect is apparently mediated through small G proteins facilitating the release of pro-inflammatory cytokines.

Oxidative stress and macrophage function : a failure to resolve the inflammatory response

The suppression of pro-inflammatory gene expression along with the clearance of apoptotic cells by phagocytosis can play an important role in resolving the inflammatory response. Any impairment of these processes can therefore lead to a chronic inflammatory state. Oxidative stress can have both direct and indirect effects on macrophage function. This mini-review highlights a mechanism through which oxidative stress via the production of reactive carbonyls alters the ECM (extracellular matrix) environment of macrophages, thereby altering their behaviour. Carbonyl modification of ECM proteins causes increased macrophage adhesion and activation through receptors that are also involved in phagocytosis. Moreover, interaction of macrophages with these carbonyl-modified ECM proteins leads to decreased phagocytic activity towards apoptotic cells. At a more direct level, both oxidative and carbonyl stress inhibits activity of the transcriptional co-repressor HDAC-2 (histone deacetylase 2), which under normoxic conditions helps to suppress pro-inflammatory gene expression. Consequently, macrophages activated under conditions of oxidative or carbonyl stress can lead to a more enhanced inflammatory response. Coupled with an impairment of the phagocytic response, this can lead to ineffective clearance of apoptotic cells and secondary necrosis, with the result being failure to resolve the inflammatory response and the establishment of a chronic inflammatory state.

Cigarette Smoke-Induced Oxidative Stress and TGF-β1 Increase p21waf1/cip1 Expression in Alveolar Epithelial Cells

Abstract: Sustained oxidative stress caused by cigarette smoking induces a chronic inflammatory response, resulting in the destruction of the alveolar cell wall characteristic of emphysema. The loss of tissue may involve the progressive depletion of epithelial cells through inhibition of proliferation leading to cell death. The cell cycle regulator p21waf1/cip1 acts as a G1/S and G2/M phase checkpoint regulator. We hypothesize that cigarette smoke‐induced oxidative stress and transforming growth factor beta 1 (TGF‐β1) may inhibit cellular proliferation by p21waf1/cip1 in type II alveolar epithelial cells (A549). A significant increase was observed in p21waf1/cip1 mRNA expression in A549 cells by cigarette smoke condensate, H2O2, and TGF‐β1. In conclusion, cigarette smoke‐induced oxidative stress and TGF‐β1 modulate expression of the cell cycle inhibitor p21waf1/cip1. This may be important in the growth arrest and cell survival of alveolar type II cells in the G1 phase.

A model of chronic inflammation and pulmonary emphysema after multiple ozone exposures in mice

Oxidative stress plays a role in the pathophysiology of emphysema through the activation of tissue proteases and apoptosis. We examined the effects of ozone exposure by exposing BALB/c mice to either a single 3-h exposure or multiple exposures over 3 or 6 wk, with two 3-h exposures per week. Compared with air-exposed mice, the increase in neutrophils in bronchoalveolar lavage fluid and lung inflammation index was greatest in mice exposed for 3 and 6 wk. Lung volumes were increased in 3- and 6-wk-exposed mice but not in single-exposed. Alveolar space and mean linear intercept were increased in 6- but not 3-wk-exposed mice. Caspase-3 and apoptosis protease activating factor-1 immunoreactivity was increased in the airway and alveolar epithelium and macrophages of 3- and 6-wk-exposed mice. Interleukin-13, keratinocyte chemoattractant, caspase-3, and IFN-γ mRNA were increased in the 6-wk-exposed group, but heme oxygenase-1 (HO-1) mRNA decreased. matrix metalloproteinase-12 (MMP-12) and caspase-3 protein expression increased in lungs of 6-wk-exposed mice. Collagen area increased and epithelial area decreased in airway wall at 3- and 6-wk exposure. Exposure of mice to ozone for 6 wk induced a chronic inflammatory process, with alveolar enlargement and damage linked to epithelial apoptosis and increased protease expression.

Oxidative stress and steroid resistance in asthma and COPD: pharmacological manipulation of HDAC-2 as a therapeutic strategy

Insensitivity to corticosteroid treatment in inflammatory conditions, such as asthma and chronic obstructive pulmonary disease, present considerable management problems and cost burdens to health services. Oxidative stress is a major component of chronic inflammation and can have a significant suppressive effect on corticosteroid efficacy. Recent advances in the understanding of both the mechanisms of corticosteroid action and corticosteroid insensitivity have provided hope for a therapeutic strategy of restoring corticosteroid sensitivity. Histone deacetylase 2 (HDAC-2) plays a pivotal role in corticosteroid action and is reduced in many cases of steroid insensitivity. Moreover, it has shown that oxidative stress can be responsible for this reduction in HDAC-2 activity. Two structurally different compounds; methyl-xanthine theophylline and polyphenol curcumin restore HDAC activity, thereby restoring corticosteroid function. Low, subbronchodilator doses of theophylline can also act as corticosteroid-sparing drugs in asthmatics. Although these compounds appear to restore corticosteroid function and may initially provide therapeutic potential, they lack specificity and the mechanism of their action is unknown. Once their mechanisms of action are established, it is likely that derivatives of these compounds may be used as a therapeutic strategy to restore corticosteroid insensitivity in the future.

Pharmacological and dietary antioxidant therapies for chronic obstructive pulmonary disease.

The progression and exacerbations of chronic obstructive pulmonary disease (COPD) are intimately associated with tobacco smoke/biomass fuel-induced oxidative and aldehyde/carbonyl stress. Alterations in redox signaling proinflammatory kinases and transcription factors, steroid resistance, unfolded protein response, mucus hypersecretion, extracellular matrix remodeling, autophagy/apoptosis, epigenetic changes, cellular senescence/aging, endothelial dysfunction, autoimmunity, and skeletal muscle dysfunction are some of the pathological hallmarks of COPD. In light of the above it would be prudent to target systemic and local oxidative stress with agents that can modulate the antioxidants/ redox system or by boosting the endogenous levels of antioxidants for the treatment and management of COPD. Identification of various antioxidant agents, such as thiol molecules (glutathione and mucolytic drugs, such as N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine, ergothioneine, and carbocysteine lysine salt), dietary natural product-derived polyphenols and other compounds (curcumin, resveratrol, green tea catechins, quercetin sulforaphane, lycopene, acai, alpha-lipoic acid, tocotrienols, and apocynin) have made it possible to modulate various biochemical aspects of COPD. Various researches and clinical trials have revealed that these antioxidants can detoxify free radicals and oxidants, control expression of redox and glutathione biosynthesis genes, chromatin remodeling, and ultimately inflammatory gene expression. In addition, modulation of cigarette smoke-induced oxidative stress and related cellular changes have also been reported to be effected by synthetic molecules. This includes specific spin traps like α-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a superoxide dismutase mimetic M40419, lipid peroxidation and protein carbonylation blockers/inhibitors, such as edaravone and lazaroids/tirilazad, myeloperoxidase inhibitors, as well as specialized pro-resolving mediators/inflammatory resolving lipid mediators, omega-3 fatty acids, vitamin D, and hydrogen sulfide. According to various studies it appears that the administration of multiple antioxidants could be a more effective mode used in the treatment of COPD. In this review, various pharmacological and dietary approaches to enhance lung antioxidant levels and beneficial effects of antioxidant therapeutics in treating or intervening the progression of COPD have been discussed.